Understanding Rice- Magnaporthe Oryzae Interaction in Resistant and Susceptible Cultivars of Rice under Panicle Blast Infection Using a Time-Course Transcriptome Analysis

Abstract

Rice blast is a global threat to food security with up to 50% yield losses. Panicle blast is a more severe form of rice blast and the response of rice plant to leaf and panicle blast is distinct in different genotypes. To understand the specific response of rice in panicle blast, transcriptome analysis of blast resistant cultivar Tetep, and susceptible cultivar HP2216 was carried out using RNA-Seq approach after 48, 72 and 96 h of infection with Magnaporthe oryzae along with mock inoculation. Transcriptome data analysis of infected panicle tissues revealed that 3553 genes differentially expressed in HP2216 and 2491 genes in Tetep, which must be the responsible factor behind the differential disease response. The defense responsive genes are involved mainly in defense pathways namely, hormonal regulation, synthesis of reactive oxygen species, secondary metabolites and cell wall modification. The common differentially expressed genes in both the cultivars were defense responsive transcription factors, NBS-LRR genes, kinases, pathogenesis related genes and peroxidases. In Tetep, cell wall strengthening pathway represented by PMR5, dirigent, tubulin, cell wall proteins, chitinases, and proteases was found to be specifically enriched. Additionally, many novel genes having DOMON, VWF, and PCaP1 domains which are specific to cell membrane were highly expressed only in Tetep post infection, suggesting their role in panicle blast resistance. Thus, our study shows that panicle blast resistance is a complex phenomenon contributed by early defense response through ROS production and detoxification, MAPK and LRR signaling, accumulation of antimicrobial compounds and secondary metabolites, and cell wall strengthening to prevent the entry and spread of the fungi. The present investigation provided valuable candidate genes that can unravel the mechanisms of panicle blast resistance and help in the rice blast breeding program.

Keywords: Magnaporthe; RNA-Seq; cell wall modification; disease resistance; panicle blast; rice.

Figure 1

Inoculation of M. oryzae to rice panicles and disease phenotyping. (A) Suspension of M. oryzae spores, ready to infect the host. (B) Infection was given to the neck of rice panicle using 5 mL syringe. (C) Representative image of rice panicles showing the impact of pathogen on blast resistant (Tetep) and susceptible (HP2216) cultivars at different time intervals at 48 hpi, 72 hpi and 96 hpi (hpi= hour post infection) along with mock inoculated.

Figure 2

Differentially expressed genes in Tetep and HP2216. Study of plant-pathogen interaction using Venn and expression profiling in Tetep and HP2216 rice, infected with M. oryzae, at different time intervals. (A) Total expressed loci common and unique between Tetep and HP2216 in mock samples without infection (control). (B) Significant (FDR adjusted p ≤ 0.01) DELs (log2 fold change ≥2) common and unique in Tetep and HP2216 at 48 hpi. (C) Significant DELs common and unique in Tetep and HP2216 at 72 hpi. (D) Significant DELs common and unique in Tetep and HP2216 at 96 hpi. (E) Significant DELs common and unique between Tetep and HP2216 at all three-time intervals. (F) Heat map of significant (FDR adjusted p ≤ 0.05 & log2 fold change ≥2) DELs of Tetep and their respective log2 fold change in HP2216. Red represents up-regulated loci and green represents down-regulated loci.

Figure 3

Gene Ontology for significant differentially expressed genes. Graphical representation of significant differentially expressed loci (SDEL; FDR adjusted p ≤ 0.01) in HP2216 comparative to Tetep present in different biological processes, molecular functions and cellular components of all significant GO terms at all three-time intervals. (A) Significant GO terms in HP2216 comparative to Tetep at 48 hpi. (B) Significant GO terms in HP2216 as comparative to Tetep at 72 hpi. (C) Significant GO terms in HP2216 comparative to Tetep at 96 hpi. GO terms for biological processes are biological regulation (GO:0065007), cellular metabolic process (GO:0044237), cellular process (GO:0009987), localization (GO:0051179), metabolic process (GO:0008152), nitrogen compound metabolic process (GO:0006807), post-translational modification (GO:0043687), response to stimulus (GO:0050896) and transport (GO:0006810). GO terms of molecular function are binding (GO:0005488), catalytic activity (GO:0003824), kinase activity (GO:0016301), transcription regulator activity (GO:0030528), transferase activity (GO:0016740) and transmembrane transporter activity (GO:0022857). GO terms of cellular components are cell (GO:0005623), cell part (GO:0044464), membrane (GO:0016020), membrane part (GO:0044425), membrane bound organelle (GO:0043227) and organelle (GO:0043226). The green bars represent for HP2216 and blue bars represent for Tetep.

Figure 4

qRT-PCR validation of selected differentially expressed genes. qRT-PCR validation of significant differentially expressed loci (SDEL) in HP2216 and Tetep upon M. oryzae infection. The green and blue bars represent the absolute fold change of HP2216 and Tetep, respectively. 18s was used for transcript normalization. Standard error bar shows the standard deviation for three replicate assays. LOC_Os06g08360 (ethylene-responsive element-binding protein), LOC_Os06g12210 (helix-loop-helix DNA-binding domain containing protein), LOC_Os05g07820 (leucine-rich repeat receptor protein kinase EXS precursor), LOC_Os01g62480 (laccase precursor protein), LOC_Os03g46860 (helix-loop-helix DNA-binding protein), LOC_Os08g43700 (OsSAUR36 –small Auxin-responsive SAUR gene family member), LOC_Os06g09860 (expressed protein), LOC_Os03g17200 (plant-specific domain TIGR01589 family protein), LOC_Os04g56430 (cysteine-rich receptor-like protein kinase), LOC_Os02g43790 (ethylene-responsive transcription factor), LOC_Os04g59150 (peroxidase precursor), LOC_Os03g16960 (cysteine-rich repeat secretory protein 55 precursor).

Figure 5

MapMan analysis for significant differentially expressed genes. MapMan overview of significant (FDR adjusted p ≤ 0.05 & log2 fold change ≥2) differentially expressed loci (SDEL) involved in biotic stress pathway, in HP2216 and Tetep, at different time intervals of M. oryzae infection. SDEL are binned to MapMan functional categories and values represented as log2 fold change values. (A,B) MapMan overview of SDEL in biotic stress pathways in HP2216 and Tetep respectively at 48 hpi. (C,D) MapMan overview of SDEL in biotic stress pathways in HP2216 and Tetep respectively at 72 hpi. (E,F) MapMan overview of SDEL in biotic stress pathways in HP2216 and Tetep respectively at 96 hpi. Red color represents up-regulated loci and green represents down-regulated loci.ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid; HSPs, heat shock proteins; R genes, resistance genes; ERF, ethylene response factor; bZIP, basic region-leucine zipper; WRKY, WRKY family of transcription factors; MYB, myeloblastosis family of transcription factors; DOF, DNA-binding with one finger plant specific transcription factors; MAPK, mitogen-activated protein kinase; PR-protein, pathogenesis-related protein.

Figure 6

Overview of mechanism of rice blast resistance in Tetep at cellular level. A model to represent the resistance against M. oryzae infection in resistant rice cultivar Tetep. M. oryzae spore in the form of conidia attaches to the surface of tissue using a hook like structure and grows a germ tube to penetrate inside the epithelial cell layer. This stimulus triggers a cell wall precursor synthesis pathway leading to generation of two NADPH molecules and their subsequent oxidation for ROS production. This activates the kinase and hormone signaling molecules. Simultaneously, jasmonic acid production starts at the cell membrane of infected cells that triggers downstream hormone signaling. Kinase signaling molecules activate transcription factors (bZIP, MYB, WRKY). Altogether, it results in expression of defense related genes like Pox, Pal, Lac, Chit, Glc, Lpo leading to synthesis of PHY and PHO type of antimicrobial compounds that responds to M. oryzae growth on the cell wall. Abbreviations: GTP, Guanosine-5’-triphosphate; UDP, Uridine diphosphate; UTP, Uridine-5’-triphosphate; PPi, Pyrophosphate; NADP⁺, Nicotinamide adenine dinucleotide phosphate; CO2, Carbondioxide; ROS, reactive oxygen species; HR, hypersensitive response; PCD, programmed cell death; TF, transcription factor; ET, ethylene; JA, jasmonic acid; PR proteins, pathogenesis-related proteins; CA, cinnamic acid; PAL, phenylalanine ammonia lyase; Glc, glucanases; Chit, chitinases; LPO, lipoxygenases; POX, peroxidases; Lac, laccases; PHO, phenolics; PHY, phytoalexin. Symbols: 1, UDP-glycosyltransferase; 2, UDP-glucose 6-dehydrogenase.